Powdered human milk fortifier

ABSTRACT

The invention relates to a powdered human milk fortifier comprising a protein component typically present in an amount of from about 24 wt/wt % to about 55 wt/wt % of the fortifier powder, and a fat component typically present in an amount of from about 1 wt/wt % to about 30 wt/wt % of the fortifier powder. Preferably, the powdered human milk fortifier is provided in a unit dose container which holds from about 0.5 gm to about 10 gm of powder. The instant invention also relates to a method of providing nutrition to preterm infants by adding a fortifier powder to human milk and administering the fortified human milk to a premature infant. The invention further provides a method of promoting growth of a premature infant by administering fortified human milk to a premature infant.

CROSS REFERENCE

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/910,094 filed Jul. 20, 2001, now allowed; which is acontinuation of U.S. Pat. No. 6,294,206 issued Sep. 25, 2001; which is acontinuation-in-part of U.S. patent application Serial No. 60/128,575filed Apr. 9, 1999, now abandoned.

TECHNICAL FIELD

[0002] The instant invention relates to a powdered human milk fortifier.The instant invention also relates to a method of providing nutrition topreterm infants by adding the fortifier powder to human milk andadministering the fortified human milk to a premature infant. Theinvention further provides a method of promoting growth of a prematureinfant by administering the fortified human milk to a premature infant.The invention also relates to a method for enhancing the physicalstability of human milk by adding an emulsifier to the human milk.

BACKGROUND

[0003] Human milk has long been recognized as the ideal feeding for terminfants because of its nutritional composition and immunologic benefits.For these reasons, mature donor human milk was considered a desirablefeeding for preterm, low-birth-weight (LBW) infants in early newbornintensive care units (NICUs). However, mature donor milk was found notto provide enough of some nutrients to meet rapidly growing LBW infants'needs. There were also concerns about possible bacterial, viral andother contamination of donor milk. For these reasons, milk from thepremature infant's own mother has become the preferred feeding in themodern NICU.

[0004] Preterm infants are commonly fed either a commercial infantformula designed specifically for these infants or their own mother'smilk. Research is still underway regarding the nutritional requirementsof these infants. However, numerous studies have documented thatunsupplemented preterm milk and banked term milk provide inadequatequantities of several nutrients to meet the needs of these infants(Davies, D. P., “Adequacy of expressed breast milk for early growth ofpreterm infants”. ARCHIVES OF DISEASE IN CHILDHOOD, 52, p.296-301,1997). Estimated energy requirements of the growing LBW infantare approximately 120 Cal/kg/day. Exact energy needs vary among infantsbecause of differences in activity, basal energy expenditure, theefficiency of nutrient absorption, illness and the ability to utilizeenergy for tissue synthesis. At 120 Cal/kg/day, about 50% of the energyintake of an LBW infant is expended for basal metabolic needs, activityand maintenance of body temperature. About 12.5% is used to synthesizenew tissue, and 25% is stored. The remaining 12.5% is excreted. Maturepreterm human milk is estimated to contain about 67 Cal/100 ml. Toachieve an intake of 120 Cal/kg/day, a LBW infant needs to consume about180 ml of preterm milk/kg/day. This feeding volume is often not welltolerated. Volumes of 100 to 150 ml/kg/day are typically fed. Therefore,to achieve an intake of 120 Cal/kg/day in an acceptable volume, thecaloric content of preterm milk must be supplemented.

[0005] Additionally, relative to estimates of the infant's requirements,preterm human milk is lacking in calcium, phosphorus and protein. Whenpreterm human milk is fortified with protein and energy, a LBW infant'sgrowth approaches that occurring in utero. Additionally, when fortifiedwith calcium and phosphorus, there is increase accretion of theseminerals and improvement of bone density. Thus, it has been recommendedthat when preterm infants are fed preterm human milk, the human milk befortified to better meet the nutritional needs of the preterm infant.

[0006] Liquid and powder forms of preterm milk fortifiers have beenmarketed domestically in response to this recognized need. The energyand nutrient composition for a typical days supplement of commerciallyavailable powder and liquid human milk fortifiers are presented inTable 1. TABLE 1 Energy and Nutrient Composition of Human MilkFortifiers Enfamil ® Human Similac Natural Milk Fortifier Care ® LiquidNutrients Powder (3.8 g) (100 ml) Energy, Kcal 14 81 Protein , gm 0.72.2 source whey protein nonfat milk, whey concentrate, protein sodiumcaseinate concentrate Fat, gm <0.1 4.4 source none added MCT, soy,coconut Carbohydrate, gm 2.7 8.6 source corn syrup solids corn syrupsolids, lactose Minerals calcium, mg 90 171 phosphorus, mg 45 94magnesium, mg 1 10 zinc, mg 0.71 1.22 manganese, mcg 4.7 9.8 copper, mg0.06 0.2 sodium, mg 7 35 potassium, mg 15.6 105 chloride, mg 17.7 66iron, mg none added 0.3 selenium, mcg none added 1.46 Vitamins A, IU 9501,008 D, IU 210 122 E, IU 4.6 3.2 K, mcg 4.4 10 Thiamin, mg 0.15 0.2Riboflavin, mg 0.21 0.5 B6, mg 0.11 0.2 B12, mcg 0.18 0.45 Niacin, mg 34 Folic acid, mcg 25 30 Pantothenic acid, mg 0.73 1.54 Biotin, mcg 2.730 C, mg 11.6 30

[0007] Similac Natural Care® and Enfamil® Human Milk Fortifier arecommercially available human milk fortifiers. The fortifiers differ withrespect to their form, source of ingredients and energy and nutrientcomposition. There is need in the NICU for both liquid and powderedhuman milk fortifiers. Powder products are advantageous to minimize thedilution of mother's milk. For example, dilution of mother's milk isundesirable when a mom is able to produce and pump sufficient milk tomeet the volume needs of her infant, However, if mom's milk supply islimited, a liquid fortifier may be used to stretch her supply of humanmilk. Similac Natural Care® is designed to be added to preterm milk in aone-to-one ratio or fed alternately with human milk meeting a need inthe NICU.

[0008] Generally, premature infants stay in the NICU for several weeksafter their mother has been released from the hospital. These tinyinfants can easily be held in the palm of an adult hand. They areusually placed in special incubators; they are on respirators to assistin their breathing; they have several indwelling catheters foradministration and/or withdrawal of fluid samples; and are intubated fortube feeding.

[0009] The method of enteral feeding chosen for each infant is based ongestational age, birth weight, clinical condition and experience of thehospital nursing personnel. Specific feeding decisions that are made bythe clinician include age to initiate feeding, route of feedingdelivery, feeding frequency, strength of feeding, and rate ofadvancement. The route for enteral feeding is determined by the infant'sability to coordinate sucking, swallowing, and breathing, which appearat approximately 32 to 34 weeks' gestation. Preterm infants of thisgestational age who are alert and vigorous may be fed by nipple. Infantswho are less mature, weak, or critically ill require feeding by tube toavoid the risk of aspiration and to conserve energy. Nasogastric andorogastric feedings, the most commonly used tube feedings in theneonatal intensive care unit, may be accomplished with bolus orcontinuous infusions of fortified human milk. Infants who receivenasogastric or orogastric feedings may be fed on an intermittent bolusor continuous schedule. Intermittent feedings every 2 to 3 hourssimulate the pattern of feeding the infant will have when advanced tobottle feeding or breast feeding. Continuous feedings may be bettertolerated by very small infants, infants who previously have nottolerated bolus feedings and infants in whom clinically significantmalabsorption develops with bolus feedings. However, reduced nutrientdelivery is a problem associated with continuous feeding. Fat from humanmilk tends to adhere to the feeding tube surfaces and reduce energydensity. Likewise, the loss of nutrients for fortifiers used tosupplement human milk is increased when given in a continuous feeding.

[0010] In order to continue feeding the infants their own mother's milkafter their mother's discharge from the hospital, the mother mustexpress milk at home into suitable containers, store the milk in therefrigerator or freezer and transport the expressed milk to the NICU.Once at the NICU, the milk is stored in refrigerator or freezertemperatures depending on the milk volume required for feeding that day.Typically, the amount of milk that will be fed to the infant within 24hours after being expressed is refrigerated. The extra expressed milk isfrozen. Consequently, the expressed milk may be subjected to severaldifferent storage conditions prior to preparation as a days feeding.

[0011] Human milk fortification is generally used for all infants whorequire tube feeding of human milk and for a few infants who requirefluid restriction. Typical feeding protocols for premature infants(<1500 g) include the addition of fortifier once the infant is receivingunfortified human milk at approximately 100 ml/kg/day. The fortifier isadded at half dose initially. For example, two 0.96 gm packets ofEnfamil® Human Milk Fortifier is added to 100 ml of mother's milk. Ifthe infant tolerates the fortified milk for 24 hours, the fortifier isincreased to full dose. In the case of the example above, the fortifieris increased to four 0.96 gm packets in 100 ml of mother's milk.

[0012] Typically, the amount of human milk prepared is based on theamount of milk needed to provide the infant with a 24-hour supply. Forexample, a 1500 gm infant would be fed 150 ml of milk a day. If frozenmilk is used, the frozen milk is placed in a warm water bath untilcompletely thawed. Special attention is given to mixing in thefortifiers. Gentle mixing is required to avoid breaking the milk fatglobule, which can increase the adherence of the milk fat to the sidesof feeding containers and result in significant loss of fat (energy).The prescribed amount of fortified milk is drawn up into syringes andlabeled with identification. When milk preparation is complete, thelabeled, aliquoted feedings are delivered to the nurseries and placedinto refrigerators for easy access by the nursing staff. Typically, therefrigerated fortified milk is warmed prior to feeding. For example, thefortified milk is warmed in a dry heat laboratory incubator set within arange of 35-45° C. for a maximum of 15 minutes. This brings thetemperature of the fortified milk to room temperature. The fortifiedmilk may be administered to the infant as a bolus feeding or through asyringe infusion pump for continuous feeding. If an infusion pump isused, the syringe tip is positioned upright to allow for a continuousinfusion of fat and the syringe is attached directly to the feeding tubeto decrease the potential surface area that the fat and immunologiccomponents may adhere to. The primary advantage of the powderedfortifier is that there is minimal dilution of human milk. There iscurrently only one powdered human milk fortifier available on thedomestic market (Enfamil® Human Milk Fortifier). Four packets ofEnfamil® Human Milk Fortifier powder (0.96 g powder/packet) is added to100 mL of preterm milk. A study in preterm infants receiving thispowdered fortifier demonstrated poor fat absorption (Schanler,“Suitability of human milk for the low-birth weight infant”, CLINICS INPERINATOLOGY, 22, pp. 207-222, 1995). Poor fat absorption negativelyimpacts the growth in these premature infants. In addition, reports fromthe NICUs described a residue that clung to the walls of thereconstitution container when the commercially available fortifierpowder was added to human milk and there were concerns that the infantswere not actually receiving all the nutrients in the fortified milk.

[0013] There is a need for a powdered human milk fortifier which is welltolerated by preterm infants and which demonstrates good fat absorptionto provide much needed energy to the preterm infant. Additionally, thereis a need for a powdered human milk fortifier that reconstitutes well inhuman milk so that all of the nutrients are actually delivered to thepreterm infant. Further, there is a need for a method of preventing thehuman milk emulsion from breaking and causing the fat to cling to thesyringe and feeding tube thereby under delivering much needed energycalories.

[0014] The instant invention is a powdered human milk fortifier thatpromotes the physical stability of the fortified human milk admixture.Further, the powdered human milk fortifier of the instant invention iswell tolerated and maximizes the health benefits of human milk whileaddressing the variability of human milk as a sole source of energy,protein, calcium, phosphorus, sodium and other micronutrients.

SUMMARY OF THE INVENTION

[0015] Relative to estimates of the low birth weight (LBW) infant'srequirements, human preterm human milk is lacking in calcium,phosphorus, energy and protein. When preterm human milk is fortifiedwith protein and energy, a LBW infant's growth approaches that occurringin utero. Additionally; when fortified with calcium and phosphorus,there is increase accretion of these minerals and improvement of bonedensity. Thus, it has been recommended that when preterm infants are fedpreterm human milk, the human milk be fortified to better meet thenutritional needs of the preterm infant.

[0016] The fortifier powder of the instant invention improves upon theprior art fortifier powder by providing higher amounts of protein andfat in the fortifier powder thereby improving the growth patterns ofpreterm infants when compared to infants fed the prior art fortifierpowder. Protein precipitation issues of the prior art fortifier powderhave also been successfully addressed through the addition of insolublecalcium which surprisingly did not negatively impact the bonedevelopment of these premature infants. Further, the addition of thesmall amount of emulsifier in the fortifier powder of the instantinvention to human milk surprisingly improved the emulsion stability ofthe fortified human milk.

[0017] The instant invention generally relates to a powdered human milkfortifier which typically includes a protein component present in aquantity of from about 24 wt/wt % to about 55 wt/wt % of the fortifierpowder, a fat component present in a quantity of from about 1 wt/wt % toabout 30 wt/wt % of the fortifier powder and a carbohydrate componentpresent in a quantity of from about 15 wt/wt % to about 75 wt/wt % ofthe fortifier powder.

[0018] Milk fat globules of human milk are known to separate from themilk and adhere to the sides of feeding containers which result insignificant loss of fat, a major source of energy for the infant. Anemulsifier not only helped the water soluble and insoluble components ofthe fortifier powder to incorporate into the human milk, it surprisinglyhelped to prevent phase separation of expressed human milk. Typically,an emulsifier is present in the fortifier powder in a quantity of fromabout 1 wt/wt % to about 10 wt/wt % of the fat component whichcorresponds with 0.1 wt/wt % to about 1 wt/wt % of the fortifier powder.

[0019] Small volumes of fortified human milk (25 ml to 100 ml) areprepared for a days feeding of a premature infant. Consequently, a bulkcontainer of fortifier powder would be repeatedly opened, powder scoopedout, recovered and stored which generates concerns about powdersterility in a hospital environment. Individual unit doses allow foraddition of small amounts of powder to human milk without thepossibility of contamination of the remaining powder since all of thepowder is used in a single preparation. Preferably, the fortifier powderis provided in individual unit dose containers which holds from about0.5 gm to about 10 gm of the fortifier powder.

[0020] The instant invention also relates to a method of providingnutrition to preterm infants by adding the fortifier powder to humanmilk and administering the fortified human milk to a premature infant.The invention further provides a method of promoting growth of apremature infant by administering the fortified human milk to apremature infant.

[0021] The invention also relates to a method for enhancing the emulsionstability of human milk by adding an emulsifier to the human milk.

DETAILED DESCRIPTION OF THE INVENTION

[0022] As used herein:

[0023] The terms “premature”, “preterm” and “low-birth-weight (LBW)”infants are used interchangeably and refer to infants born less than 37weeks gestational age and/or with birth weights less than 2500 gm.

[0024] “A unit dose” refers to individual packages of fortifier powdercontaining an amount of fortifier powder that will be used in apreparation. There will be no leftover fortifier powder requiringstorage. The amount of fortified human milk prepared for a prematureinfant typically ranges from 25 ml to 150 ml a day. Consequently, asingle unit dose is the appropriate amount of powder to fortify a 25 mlpreparation. Multiple packages are added to the larger volumepreparations.

[0025] The term “growth” refers to gains in weight, length and/or headcircumference.

[0026] The term “insoluble calcium” refers to food grade calcium sourceslisted in the CRC HANDBOOK OF CHEMISTRY AND PHYSICS as sparingly solublein water.

[0027] The term “vitamin E” refers to a group of tocopherols that differonly in the number and position of methyl groups on the ring. The mostactive form of vitamin E, is also the most widely distributed in nature.When tocopherol was first synthesized, the synthetic material was foundto have a slightly lower biological activity than the tocopherol fromplants. Because of this phenomena, the natural occurring form has beendesignated RRR-“-tocopherol. For dietary purposes, vitamin E activity isexpressed as RRR-”-tocopherol equivalents (TEs). One TE is the activityof 1 mg of RRR-“-tocopherol. One mg of RRR-”-tocopherol is equivalent to1.49 IU of vitamin E.

[0028] “Maltodextrins” and “corn syrups” refer to complex carbohydratesroutinely used in nutritional formulations because of their excellentdigestibility and functional properties. Specifically, they are goodwater binders and provide products with desired texture and mouth feel.Maltodextrins are polysaccharides obtained from the acid or enzymehydrolysis of corn starch. Their classification is based on the degreeof hydrolysis and is reported as “dextrose equivalence (DE)”. The FDAdefines maltodextrins as non-sweet, nutritive polysaccharides that havea DE less than 20. Corn syrup solids are defined as having DE's greaterthan 20. Corn syrup solids consist of dextrose chains about 3 to 4 unitslong while maltodextrins are less hydrolyzed and contain longer dextrosechains. The difference in polymer length results in differentfunctionality, viscosity, mouth feel and osmolality.

[0029] It is a principal object of the invention to provide an improvedpowdered human milk fortifier for premature infants who requireadditional nutrients to support their growth. The invention is a powderwhich when added to human milk supplements the levels of protein, fat,vitamins and minerals. Another object of this invention is to provide amethod for providing supplemental nutrients to a premature infant whorequires additional nutrients for growth.

[0030] Although not intended to limit the invention in any manner, butto merely serve as a general guideline, the fortifier powder of thisinvention will typically provide the following macronutrientdistribution. The protein component will typically be present in anamount of from about 24 wt/wt % to about 55 wt/wt % of the fortifierpowder, preferably from about 25 wt/wt % to about 42 wt/wt % of thefortifier powder, more preferably from about 28 wt/wt % to about 36wt/wt % of the fortifier powder. The fat component will typically bepresent in an amount of from about 1 wt/wt % to about 30 wt/wt % of thefortifier powder, preferably from about 5 wt/wt % to about 20 wt/wt % ofthe fortifier powder, more preferably from about 8 wt/wt % to about 18wt/wt % of the fortifier powder. The carbohydrate component willtypically be present in an amount of from about 15 wt/wt % to about 75wt/wt % of the fortifier powder, preferably from about 38 wt/wt % toabout 70 wt/wt % of the fortifier powder, more preferably from about 46wt/wt % to about 64 wt/wt % of the fortifier powder. Additionally, theamount of powder required to provide a unit dose of the fortifier willtypically range from about 0.5 gm to about 10 gm of powder in a unitdose, preferably from about 0.8 gm to about 5.0 gm of powder in a unitdose, more preferably from about 0.85 gm to about 2.0 gm of powder in aunit dose. The caloric density is typically from about 1.0 kcal/gmpowder to about 8.5 kcal/gm powder.

[0031] The first component of the fortifier powder of this invention isa source of protein. Protein is needed for growth, synthesis of enzymesand hormones, and replacement of protein lost from the skin and in urineand feces. These metabolic processes determine the need for both thetotal amount of protein in a feeding and the relative amounts ofspecific amino acids. The adequacy of the amount and type of protein ina feeding for infants is determined by measuring growth, nitrogenabsorption and retention, plasma amino acids, certain blood analytes andmetabolic responses.

[0032] As stated above, the protein component will typically be presentin an amount of from about 24 wt/wt % to about 55 wt/wt % of thefortifier powder. The proteins that may be utilized in the nutritionalproducts of the invention include any proteins or nitrogen sourcesuitable for human consumption. Such proteins are well known by thoseskilled in the art and can be readily selected when preparing suchproducts. Examples of suitable protein sources for a premature infanttypically include casein, whey, condensed skim milk, nonfat milk, soy,pea, rice, corn, hydrolyzed protein, free amino acids, protein sourceswhich contain calcium in a colloidal suspension with the protein andmixtures thereof.

[0033] The preferred protein source will typically be comprised of about51 wt/wt % of the protein component as whey protein concentrate andabout 49 wt/wt % of the protein component as nonfat dry milk whichcorresponds to about 60 wt/wt % of the protein component as whey andabout 40 wt/wt % of the protein component as casein.

[0034] Commercial protein sources are readily available and known to onepracticing the art. For example, caseinates, whey, hydrolyzedcaseinates, hydrolyzed whey and milk proteins are available from NewZealand Milk Products of Santa Rosa, Calif. Soy and hydrolyzed soyproteins are available from Protein Technologies International of SaintLouis, Mo. Pea protein is available from Feinkost Ingredients Company ofLodi, Ohio. Rice protein is available from California Natural Productsof Lathrop, Calif. Corn protein is available from EnerGenetics Inc. ofKeokuk, Iowa. Additionally, mineral enriched proteins are available fromNew Zealand Milk Products of Santa Rosa, Calif. and Protein TechnologiesInternational of Saint Louis, Mo.

[0035] The second component of the fortifier powder of this invention isa source of fat. Fat is an ideal source of energy for LBW infants, notonly because of its high caloric density but also because of its lowosmotic activity in solution.

[0036] As stated above, the fat component will typically be present inan amount of from about 1 wt/wt % to about 30 wt/wt % of the fortifierpowder. Examples of suitable fat sources typically include high oleicsafflower oil, soy oil, fractionated coconut oil (medium chaintriglycerides, MCT oil), high oleic sunflower oil, corn oil, canola oil,coconut, palm and palm kernel oils, marine oil, cottonseed oil andspecific fatty acids such as docosahexaenoic acid and arachidonic acid.

[0037] Docosahexaenoic acid (DHA) is an omega-3 fatty acid and isthought to be essential for the proper brain and vision development ofinfants because it is the most abundant long chain polyunsaturated fattyacid (PUFA) in the brain and retina. Although a metabolic pathway existsin mammals for the biosynthesis of DHA from dietary linolenic acid, thispathway is bioenergetically unfavorable and mammals are thought toobtain most of their DHA from dietary sources. In the case of infants,the most likely source would be human milk. Indeed, DHA is the mostabundant 20 carbon omega-3 PUFA in human milk. However, human milk DHAcontent will vary greatly depending on the diet of the mother. If themother eats fish high in DHA often, her milk will contain higher DHAlevels, while a mom with less access to fish will have lower DHA levelsin her milk. Consequently, human milk may require DHA supplementation toinsure that the preterm infant is receiving sufficient amounts of DHA.Preferably, DHA supplementation is accompanied by arachidonic acidsupplementation. U.S. Pat. No. 5,492,938 to Kyle et al. describes amethod of obtaining DHA from dinoflagellates and its use inpharmaceutical composition and dietary supplements.

[0038] Typically, MCT oil is the preferred fat source which comprises100% of the fat component This fat source at this level provides welltolerated fat calories to the premature infant in addition to providinga vehicle for fat soluble vitamins and emulsifiers.

[0039] Numerous commercial sources for the fats listed above are readilyavailable and known to one practicing the art. For example, soy andcanola oils are available from Archer Daniels Midland of Decatur, Ill.Corn, coconut, palm and palm kernel oils are available from PremierEdible Oils Corporation of Portland, Organ. Fractionated coconut oil isavailable from Henkel Corporation of LaGrange, Ill. High oleic safflowerand high oleic sunflower oils are available from SVO Specialty Productsof Eastlake, Ohio. Marine oil is available from Mochida International ofTokyo, Japan. Sunflower and cottonseed oils are available from Cargil ofMinneapolis, Minn. Safflower oil is available from California OilsCorporation of Richmond, Calif. DHA is available from Martek BiosciencesCorporation of Columbia, Md. Arachidonic acid is available from GenzymeCorporation of Cambridge, Mass.

[0040] An emulsifier is typically incorporated into the fortifierpowder. Emulsifiers help the water soluble and insoluble components ofthe fortifier powder incorporate into the human milk. Examples ofsuitable emulsifiers typically include soya bean lecithin,polyoxythylene stearate, polyoxyethylene sorbitan mono-oleate,polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitanmonostearate, ammonium phosphatides, polyoxyethylene sorbitanmonolaurate, citric acid esters of mono and diglycerides of fatty acids,tartaric acid esters of mono and diglycerides of fatty acids.

[0041] The preferred emulsifier source is natural soy lecithin. Theamount of emulsifier will typically be present in an amount of fromabout 1 wt/wt % to about 10 wt/wt % of the fat component whichcorresponds to about 0.1 wt/wt % to about 1 wt/wt % of the fortifierpowder. Preferably the emulsifier is present in an amount of from about1.5 wt/wt % to about 5 wt/wt % of the fat component.

[0042] Numerous commercial sources for the emulsifiers listed above arereadily available and known to one practicing the art. For example, soyabean lecithin is-available from Archer Daniels Midland Company inDecatur, Ill. Polyoxythylene stearate, polyoxyethylene sorbitanmono-oleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylenesorbitan monostearate, polyoxyethylene sorbitan monolaurate, citric acidesters of mono and diglycerides of fatty acids, and tartaric acid estersof mono and diglycerides of fatty acids are available from Quest inOwings Mills, Md.

[0043] The third component of the fortifier powder of this invention isa source of carbohydrates. Carbohydrate is a major source of readilyavailable energy that the LBW infant needs for growth and that protectsthe infant from tissue catabolism. In human milk and most standardmilk-based infant formulas, the carbohydrate is lactose. LBW infants maybe unable to fully digest lactose because lactase activity in the fetalintestine is not fully developed until late in gestation (36 to 40weeks). On the other hand, sucrase activity is maximal by 32 weeks'gestation, and glucosoamylase activity, which digests corn syrup solids(glucose polymers), increase twice as rapidly as lactase activity duringthe third trimester.

[0044] As noted above, the carbohydrates will typically be present in anamount of from about 15 wt/wt % to about 75 wt/wt % of the fortifierpowder. The preferred carbohydrate level and source is selected todecrease osmolality and viscosity of the reconstituted product. Thepreferred carbohydrate source is 100% of the carbohydrate component ascorn syrup.

[0045] The carbohydrates that may be used in the fortifier powder canvary widely. Examples of carbohydrates suitable for preterm infantstypically include hydrolyzed corn starch, maltodextrin, glucosepolymers, sucrose, corn syrup, corn syrup solids, rice syrup, glucose,fructose, lactose, high fructose corn syrup and indigestibleoligosaccharides such as fructooligosaccharides (FOS). Any singlecarbohydrate listed above, or any combination thereof, as appropriatemay be utilized. Commercial sources for the carbohydrates listed aboveare readily available and known to one practicing the art. For example,corn syrup solids are available from Cerestar USA, Inc in Hammond, Ind.Glucose and rice based syrups are available from California NaturalProducts in Lathrop, Calif. Various corn syrups and high fructose cornsyrups are available from Cargil in Minneapolis, Minn. Fructose isavailable from A. E. Staley in Decatur, Ill. Maltodextrin, glucosepolymers, hydrolyzed corn starch are available from American MaizeProducts in Hammond, Ind. Sucrose is available from Domino Sugar Corp.in New York, N.Y. Lactose is available from Foremost in Baraboo, Wis.and indigestible oligosaccharides such as FOS are available from GoldenTechnologies Company of Golden, Colo.

[0046] The osmolality of the fortified human milk plays an importantrole in the infants tolerance of their feedings such as abdominaldistention and vomiting/spit-up. Osmolality of the fortified human milkis tied to the level and source of carbohydrate utilized in thefortifier powder. The osmolality of the fortifier powder of the instantinvention reconstituted in human milk is typically less than about 400mOsm/kg water, preferably from about 300 mOsm/kg water to about 400mOsm/kg water. The substitution of fat for some of the carbohydrate inthe fortifier powder of the instant invention serves to reduce theosmolality of fortified human milk by replacing the carbohydrate whichhas a high osmotic activity with fat which has a low osmotic activity.The type of carbohydrate incorporated into the fortifier powder alsoimpacts the osmolality of the fortified human milk. The more hydrolyzedthe carbohydrate source (higher DE) the higher the osmotic activity.Additionally, partially hydrolyzed carbohydrate sources may furtherincrease the osmolality when reconstituted with human milk due tofurther hydrolysis by human milk amylase. Based on the DE values forcarbohydrates, one skilled in the art can readily select thecarbohydrate source or combination of carbohydrates that will result inthe preferred osmolality of the reconstituted fortifier powder/humanmilk solution.

[0047] As stated above, viscosity is also a characteristic ofcarbohydrates. Viscosity of the reconstituted fortifier powder/humanmilk solution plays a role in suspending the insoluble minerals duringfeeding. While higher viscosity's tend to reduce insoluble mineralfallout, the higher viscosity can cause tube/nipple clogging. A cloggedfeeding tube in a continuous feeding apparatus requires additionalattention by the nursing staff who will have to unclog the tube, resetthe pump system which may require a new preparation of fortified humanmilk. More importantly, a clogged tube prevents the timely delivery ofmuch needed nutrients to a premature infant. The viscosity of thereconstituted fortifier powder/human milk solution of the instantinvention is typically less than about 30 cps, preferably from about 10cps to about 20 cps. Viscosity is inversely related to osmolality. Themore hydrolyzed a starch is (higher DE), the lower the viscosity and thehigher the osmolality. Based on the DE values for carbohydrates, oneskilled in the art can readily select the carbohydrate source orcombination of carbohydrates that will drive the viscosity andosmolality characteristics of the reconstituted fortifier powder/humanmilk solution to the preferred levels.

[0048] The fourth component of the fortifier powder of the presentinvention typically includes supplemented vitamins and minerals.

[0049] The preterm infant requires the electrolytes sodium, potassiumand chloride for growth and for acid-base balance. Sufficient intakes ofthese electrolytes are also needed for replacement of losses in theurine and stool and from the skin. Calcium, phosphorus and magnesium areneeded for proper bone mineralization. For bones to grow, adequateamounts of these minerals must be present in a feeding. LBW infants maydevelop rickets or osteopenia if they do not receive adequate amount ofcalcium and phosphorus in their diet. Phosphorus and magnesium are alsofound in intracellular fluid. These minerals are needed for the growthand function of soft tissue. Human milk does not provide enough calciumor phosphorus, even if these minerals were to be totally absorbed andretained, which they are not.

[0050] Trace minerals are associated with cell division, immune functionand growth. Consequently, provision of sufficient amounts of traceminerals is needed for rapid growth in LBW infants. Human milk does notprovide sufficient amounts of the trace minerals, especially zinc andcopper, to meet the needs of a growing LBW infant. Another tracemineral, iron, is important for the synthesis of hemoglobin, myoglobinand iron-containing enzymes. However, it is not certain that LBW infantsneed the recommended amounts of iron during the first 2 months of life.The anemia of prematurity occurring shortly after birth cannot beavoided by giving supplemental iron. Also, the preterm infant isestimated to have sufficient iron stores without receiving ironsupplementation, if blood loss is small, until 2 months of age.Consequently, the powdered human milk fortifier of the instant inventionis low in iron. Zinc is needed for growth, for the activity of numerousenzymes, and for DNA, RNA and protein synthesis. Copper is necessary forthe activity of several important enzymes. It is estimated that about75% of the copper in a term neonate is accumulated during the last 10 to12 weeks in utero. Consequently, LBW infants, especially those bornweighing less than 1500 gm, are likely to have low copper stores.Manganese is needed for the development of bone and cartilage and isimportant in the synthesis of polysaccharides and glyoproteins.

[0051] LBW infants are likely to need more of most vitamins thanprovided by human milk alone because of low vitamin stores at birth, lowintake of feedings, poor absorption of vitamins and clinical conditionsrequiring increased vitamin intakes.

[0052] Vitamin A is a fat-soluble vitamin essential for growth, celldifferentiation, vision and the immune system. The vitamin A stores inLBW infants are adequate shortly after birth but decrease soonthereafter. Therefore, preterm infants may require higher intakes ofvitamin A than term infants. Vitamin D is important for absorption ofcalcium and to a lesser extent, phosphorus, and for the development ofbone. For many years it was thought that poor bone development observedin LBW infants was due to insufficient vitamin D intake and metabolismand the LBW infants required significantly greater vitamin D intake thanterm infants. It is now recognized that calcium and phosphorus intakesare more important than vitamin D for bone growth in preterm infants.Vitamin E (tocopherol) prevents peroxidation of polyunsaturated fattyacids in the cell, thus preventing tissue damage. LBW infants maydevelop hemolytic anemia and vitamin E deficiency when fed feedings lowin vitamin E and high in iron and polyunsaturated fatty acids.Additionally, preterm milk contains very low levels of vitamin K.

[0053] As are several other water-soluble vitamins, vitamin C is low inmature preterm milk. Folic acid is important in amino acid andnucleotide metabolism. Serum folate concentrations have been shown tofall below normal after 2 weeks of age in LBW infants with low folicacid intakes. Additionally, several B vitamins are present at lowconcentrations in preterm milk.

[0054] As described above, the variability of human milk vitamin andmineral concentrations and the increased needs of the preterm infantrequires a minimal fortification to insure that a developing prematureinfant is receiving adequate amounts of vitamins and minerals while notover fortifying and possibly causing, for example, hypercalcemia. Usingthe recommendations of the Committee on Nutrition, American Academy ofPediatrics, one skilled in the art can readily calculate how much of avitamin or-mineral source should be added to the nutritional product inorder to deliver the desired amount of a vitamin or mineral.(Nutritional needs of premature infants, PEDIATRIC NUTRITION HANDBOOK,ed. 4. Elk Grove Village, Ill. American Academy of Pediatrics, 1998, pp.55-87) Practitioners also understand that appropriate additional amountsof vitamin and mineral ingredients need to be provided to nutritionalcompositions to -compensate for some loss during processing and storageof such compositions.

[0055] Examples of supplemental vitamins and minerals in the fortifierpowder of the instant invention typically include vitamin A, vitamin B₁,vitamin B₂, vitamin B₆, vitamin B₁₂, vitamin C, vitamin D, vitamin E,vitamin K, biotin, folic acid, pantothenic acid, niacin, m-inositol,calcium, phosphorus, magnesium, zinc, manganese, copper, sodium,potassium, chloride, iron and selenium. The additional nutrientschromium, molybdenum, iodine, taurine, carnitine and choline may alsorequire supplementation. Desirably, the fortifier powder will includethe natural form of vitamin E (RRR-d-alpha-tocopherol acetate).

[0056] The inventors discovered that the solubility characteristics ofsome of the minerals, particularly calcium, in the fortifier powdernegatively impact protein stability in the fortified human milksolution. Specifically, the presence of soluble divalent minerals in thefinal product destabilizes both human milk protein and fortifier proteinwhich causes the protein to precipitate out of solution and cling to thesides of the mixing containers. Experiment II evaluates proteindenaturation by fortifier powder containing soluble calcium and thefortifier powder of the instant invention containing insoluble calcium.The inventors have tested the residue left on the sides of the mixingcontainers for protein and found the residue to consist ofimmunologically active whey and casein protein and other unidentifiedprotein. Further, the inventors were able to calculate the percent oftotal protein lost as residue clinging to the sides of the mixingcontainers for each fortifier powder. Addition of fortifier powdercontaining soluble calcium to human milk resulted in the lose of 6% ofthe total protein in the fortified human milk. Protein losses this highcan have a negative impact on the growth of preterm infants.

[0057] Conventional wisdom dictates that a soluble calcium source willmaximize mineral bioavailabilty characteristics for a growing infant.This is supported by the incorporation of soluble calcium sourcescalcium gluconate and calcium glycerophosphate in the commerciallyavailable fortifier powder, Enfamil® Human Milk Fortifier. However, itis known that soluble divalent minerals, particularly calcium, arecapable of interacting with proteins. The destabilization of proteinsresults in denatured protein that precipitates out of solution or clingsto the sides of the reconstitution/delivery container. Consequently, theprotein is not actually delivered to the infant which contribute to theslower growth rates (see Experiment III).

[0058] The current invention uses insoluble calcium sources andaddresses the resulting mineral precipitation issues first by making thefortifier a powder and secondly by using particle sizes small enough(ultra micronized) to stay in solution during feeding. Examples ofsuitable insoluble calcium sources typically include calcium phosphatedibasic, calcium phosphate tribasic and calcium carbonate and calciumcitrate. Alternatively, the calcium is in a colloidal suspension withthe protein such as calcium caseinate.

[0059] Preferably, about 95% of the total calcium is supplied by calciumphosphate tribasic and about 5% of the total calcium is supplied bycalcium citrate.

[0060] Numerous commercial sources for insoluble calcium are readilyavailable and known to one practicing the art. For example, calciumphosphate tribasic, calcium phosphate dibasic, calcium citrate areavailable from Mallinckrodt Specialty Chemicals in Charlotte, N.C.Calcium carbonate is available from Prillaman Chemical Corporation inSuffolk, Va. Calcium caseinate is available from New Zealand MilkProducts in Hamilton, New Zealand.

[0061] The incorporation of insoluble calcium in the fortifier powderleads one skilled in the nutritional arts to be concerned aboutbioavailability of the calcium for this growing premature population.Preterm human milk is already deficient in calcium and-if the fortifierpowder provides calcium in a form that is not bioavailable to thepreterm infant, growth will be negatively impacted. The inventorsperformed preliminary studies (Experiment IV) evaluating the forearmbone density of infants supplemented with the fortifier powder of theinstant invention containing insoluble calcium and compared theirforearm bone density to infants supplemented with fortifier powdercontaining soluble calcium. Surprisingly, the results show no differencein bone density. The addition of insoluble calcium sources to thefortifier powder of the instant invention prevented the denaturation ofprotein in the fortified milk and was absorbed by the premature infant.Consequently, the protein and calcium were successfully delivered to thepremature infant and the infant was able to thrive and grow.

[0062] The nutritional powder of this invention can be manufacturedusing techniques well known to those skilled in the art. Whilemanufacturing variations are certainly well known to those skilled inthe nutritional formulation arts, a few of the manufacturing techniquesare described in detail in the Examples. Generally speaking an oil blendis prepared containing all oils, any emulsifier, and the fat solublevitamins. Two more slurries (carbohydrate and protein) are preparedseparately by mixing the carbohydrate and minerals together and theprotein in water. The two slurries are then mixed together with the oilblend. The resulting mixture is homogenized, heat processed,standardized with water soluble vitamins, and dried. The resultingpowder may be milled to a specific particle size and/or aggolmerized tomodify particle size and mixability characteristics. Those skilled inthe nutritional formulation arts would also be able to dry blend theindividual starting materials and add the liquid ingredients throughagglomeration or during the dry blending step.

[0063] Individual unit dose size packages are preferred over bulkpackaging. Because of the small volumes of milk administered topremature infants over the course of a days feeding, small volumes offortified human milk are prepared. Powder sterility in a bulk containerthat has been repeatedly opened, powder scooped out, recovered andstored is always a concern in a hospital environment. Individual unitdoses allow for addition of small amounts of powder to human milkwithout the possibility of contamination of the remaining powder sinceall of the powder is used in a single preparation. As noted above, theunit dose of the invention typically is the amount of from about 0.5 gmto about 10 gm of fortifier powder in a unit dose, preferably from about0.8 gm to about 5.0 gm of powder in a unit dose, more preferably fromabout 0.85 gm to about 2.0 gm of powder in a unit dose. Depending on thevolume of a days feeding, from about 1 to about 4 unit doses will beadded to about 25 ml to about 100 ml, respectively.

[0064] Numerous types of containers are readily available and known toone practicing the art. Examples of container types typically includepackets or sachets which may be manufactured of paper, foil and plasticfilm, and foil and plastic film coated paper; and ampoules which may bemanufactured of plastic, reinforced paper and glass.

[0065] As stated above, the instant invention also relates to a methodof providing nutrition to preterm infants by adding the fortifier powderof the instant invention to human milk and administering the fortifiedhuman milk to a premature infant. The invention further provides amethod of promoting growth of a premature infant by administering thefortified human milk to a premature infant. Experiments III and IVdescribe the study protocol and better growth of premature infants withthe fortifier powder of the instant invention compared to a commerciallyavailable fortifier powder.

[0066] The invention also relates to a method for enhancing the emulsionstability of human milk by adding an emulsifier to the human milk.Surprisingly, the emulsifier helps to prevent the separation of fatglobules in human milk. The inventors discovered that the addition ofthe small amount of emulsifier in the fortifier powder to human milkimproved phase separation results. The study and results comparing thephysical stability of human milk and fortifier powder/human milk aredescribed in Experiment I. The amount of emulsifier in the fortifiedhuman milk solution will typically be present in from about 0.36 wt/vol% to about 3.6 wt/vol % of the human milk solution, preferably fromabout 0.54 wt/vol % to about 1.8 wt/vol % of the human milk solution.

MANUFACTURING EXAMPLE A

[0067] A batch of fortifier powder is manufactured by combining theappropriate ingredients to generate one carbohydrate/mineral (CHO/MIN)slurry, one oil blend and one protein in water slurry (PIW). TheCHO/MIN, oil blend and PIW slurries are mixed together to form the finalblend. The final blend is then processed with an HTST treatment. Afterstandardization, the final blend is spray dried.

[0068] Table 2 presents a bill of materials for manufacturing 8,172 kgof powdered human milk fortifier. A detailed description of itsmanufacture follows. TABLE 2 Bill of materials Ingredient AmountIngredient water 16,205 L Corn syrup solids 1603 kg Magnesium chloride96.2 kg Potassium citrate 223.8 kg Sodium citrate 6.6 kg Sodium chloride15.4 kg MCT oil 801 kg Lecithin 16.6 kg Vitamin A 2.36 kg Vitamin D359.3 g Vitamin K 27.5 g Natural Vitamin E 7.6 kg Calcium carbonate 33.1kg Tricalcium phosphate 646 kg Whey protein concentrate 1506 kg Non fatdry milk 3307 kg potassium citrate 257.2 g ferrous sulfate 3.7 kg Zincsulfate 11.1 kg Copper sulfate 1.84 kg Manganese sulfate 0.320 kg Sodiumselenate 0.001 kg Niacinamide 0.98 kg Riboflavin 1.14 kg Calciumpantothenate 4.08 kg Pyridoxine hydrochloride 0.655 kg m-inositol 9.55kg Biotin 0.0727 kg Folic acid 0.0775 kg Cyanocobalamin 0.0016 kgAscorbic acid 153.5 kg

[0069] A carbohydrate/mineral slurry is prepared by heating 2,763 litersof ingredient water to 54° C.-62° C. With agitation, the specifiedamounts of corn syrup solids (Maltrin M200 distributed by GrainProcessing Corporation, Muscatine, Iowa), magnesium chloride, sodiumchloride, sodium citrate, potassium citrate, ultra micronized tricalciumphosphate and calcium carbonate are added to the heated water. Theslurry is held under agitation at 54° C.-62° C. for not longer than sixhours until it is blended with the other slurries.

[0070] An oil blend is prepared by heating the specified amount of MCToil (distributed by Stepan, Maywood, N.J.) to 32° C.-37° C. withagitation. An emulsifier (standard fluid lecithin distributed by CentralSoya, Ft. Wayne, Ind.) is then added under agitation and allowed todissolve. Vitamin A,D,K and Natural Vitamin E (distributed by Vitamins,Inc., Chicago, Ill.) are then added to the slurry with agitation. Thecompleted oil slurry is held under moderate agitation at a temperaturefrom 26° C. to 48° C. for a period of no longer than six hours until itis blended with the other slurries.

[0071] A protein-in-water slurry is prepared by heating 9,053 liters ofingredient water to 48° C.-60° C. With agitation, the specified amountof whey protein concentrate (AMP 800 distributed by AMPC, Inc. Ames,Iowa) and nonfat dry milk is added to the heated water. The completedprotein-in-water slurry is not held but blended directly with the otherslurries.

[0072] The protein-in-water, oil blend and carbohydrate/mineral slurriesare blended together with agitation and the resultant blend ismaintained at a temperature from 51° C. to 60° C. After waiting for atleast five minutes with agitation the final blend pH is adjusted with 1N KOH to a pH from 6.45 to 6.80. The total solids of the final blend is30%. The final blend is held for no longer than two hours after the pHcheck.

[0073] After waiting for a period of not less than five minutes norgreater than two hours, the blend is subjected to deaeration,high-temperature-short-time heat treatment, and homogenization, asfollows:

[0074] A. deaerate the blend at 10-15 inches Hg;

[0075] B. emulsify the blend at 900-1100 psig in a single stagehomogenizer;

[0076] C. pass the blend through a plate/coil heater and heat the mix to71° C. to 82° C.;

[0077] D. homogenize the blend at 3900 to 4100/400 to 600 psig in adouble stage homogenizer;

[0078] E. pass the blend through a 16 second hold tube at a temperaturefrom 73° C. to 85° C.;

[0079] F. cool the blend to a temperature from 1° C. to 7° C.; and

[0080] G. store the blend at a temperature from 1° C. to 7° C.

[0081] After the above steps have been completed, appropriate analyticaltesting for quality control is conducted. Based on the analyticalresults of the quality control tests, batch corrections are made if needbe. Final blend total solids are from 29% to 31%.

[0082] A water soluble vitamin solution, ascorbic acid solution andtrace mineral solution are prepared separately and added to theprocessed blend.

[0083] The ascorbic acid solution is prepared by adding the requiredamount of ascorbic acid to 2,453 liters of 10° C. to 37° C. water withagitation.

[0084] The mineral solution is prepared by heating 321 liters ofingredient water to 37° C. to 65° C. Under agitation, add the requiredamount of potassium citrate and ferrous sulfate. Allow to agitate untilthe solution is a clear green color. Add the required amounts of zincsulfate, copper sulfate, manganese sulfate and sodium selenate to thegreen mineral solution. Agitate five minutes minimum.

[0085] The water soluble vitamin solution is prepared by heating 530liters of ingredient water to 37° C. to 65° C. The required quantitiesof niacinamide, riboflavin, calcium pantothenate, pyridoxinehydrochloride, thiamine hydrochloride, m-insitol, biotin, folic acid andcyanocobalamin are added to the heated water.

[0086] All of the ascorbic acid solution, the mineral solution and watersoluble vitamin solution is then added to the blended slurry underagitation.

[0087] The final mix is preheated through a plate heater to 71° C.-82°C. before going to a surge tank. The mix leaves the surge tank andpasses through the steam injector where it is heated to 88° C.-93° C.The mix enters the vapor-flash chamber where it is cooled to 71° C.-82°C., then pumped through an in-line 200 micro filter prior to the highpressure pump and into the dryer. The dryer settings are as follows:Nozzle pressure 3000-5000 psig Liquid flow rate 11 gpm max. Ingoing AirTemperature 160° C.-207° C. Outgoing Air Temperature 82° C.-108° C.

[0088] To control bulk density, dispersibility, particle size, moistureand physical stability, the specific spray nozzle, nozzle pressure,drying temperatures and fine reinjection parameters may vary dependingupon the drying conditions of the day. The powder passes from the dryerinto the powder cooler where the powder is cooled to below 43° C. Thecooled powder is stored in appropriate containers until being filed inindividual packets.

Experiment I

[0089] The objective of the study was to evaluate the emulsion stabilityof human milk after the addition of the fortifier powder of the instantinvention. The emulsion stability of liquids which incorporate powdersis routinely tested by a phase separation test. The test evaluates theseparation of human milk into fat-soluble and water-soluble layers. 7.2gm of fortifier powder produced as in Manufacturing Example A wasweighed into small plastic cups and covered to keep dry. Using a 250 mLgraduated cylinder, 200 ml of human milk was poured into a 500 mLbeaker. The fortifier powder was slowly added to the beaker whilestirring vigorously. Stirring continued for about 30 seconds to ensurethe powder was thoroughly hydrated. The solution was immediatelytransferred to a 250 mL graduated cylinder for phase separation test.The sample sat undisturbed for 30, 60 and 120 minutes before analysis.The sample was evaluated for any evidence of a fat layer at the top. Ahigh intensity flashlight was held against the gradated cylinder to helpdistinguish between foam and fat layer. In addition, a small spatula wasplaced inside the cylinder to push on the edge of the top layer to helpdetermine if the top layer is foam or fat layer. The fat layer was readdirectly off the graduated cylinder in ml. Table 3 lists the phaseseparation results for the human milk/fortifier powder solution and thehuman milk control after 30, 60 and 120 minutes. TABLE 3 PhaseSeparation Results Sample 30-60 min 120 min fortified human milk 1 ml 1ml human milk control 2 ml 2 ml

[0090] The fat in the human milk control sample separated and rose tothe top of the solution as demonstrated by a 2 ml fat layer. Thefortified human milk had half as much fat separate and rise to the topof the solution as demonstrated by a 1 ml fat layer. Human milk fat isknown to separate and adhere to the sides of feeding containers andresult in significant loss of fat (energy). The small amount ofemulsifier added to the fortifier powder to help the powder incorporateinto the human milk was surprisingly also able to decrease the fatseparation observed in human milk, thereby delivering more caloricallydense fat to the premature infant.

[0091] As discussed above, fat is crucial for growth of prematureinfants. The lose of fat helps explain the difference in growth observedin preterm infants supplemented with the commercially available powderedhuman milk fortifier which does not contain an emulsifier and thepowdered human milk fortifier of the instant invention which contains anemulsifier. (See Experiment III and IV)

Experiment II

[0092] Reports from the NICUs described a residue that clung to thewalls of the reconstitution container when the commercially availablefortifier powder was added to human milk and there were concerns thatthe infants were not actually receiving all the nutrients in thefortified milk. The residue was examined under the electron microscopeand was found to be protein.

[0093] The objective of the study was to quantify and identify theprotein residue left on the side of the reconstitution containers ofpowdered human milk fortifier containing soluble calcium (SC) and thepowdered human milk fortifier of the instant invention containinginsoluble calcium (IC). The powdered human milk fortifier containingsoluble calcium sample was the Enfamil® Human Milk Fortifier thecomposition described in Table 1. The powdered human milk fortifiercontaining insoluble calcium sample was the fortifier powder of theinstant invention produced as described in Manufacturing Example A.

[0094] Samples of each powdered human milk fortifier were reconstitutedby adding 0.9 g of the appropriate powder to 25 mL of 2% milk in a glassgraduated cylinder. The 2% milk control contained only 25 mL of milk,but was otherwise treated in an identical manner. Each graduatedcylinder was then covered, and given 6 vigorous shakes to facilitatepowder reconstitution. Each solution was then poured into a labeledbeaker for protein analysis. Each graduated cylinder was then invertedonto a paper towel and allowed to drain for 1 minute and then setupright. 10.0 mL of Universal Assay Buffer (PBS containing 0.1% Tween 20and 0.05% ovallbumin) was added, and the cylinder was covered. Theinside walls of the cylinder were rinsed by shaking to get particulatesoff the -side walls and into the buffer solution. To facilitatesolubilization of the residue, each cylinder was then incubated for 15minutes in a water bath at 37° C., then allowed to cool to roomtemperature prior to further dilution in buffer.

[0095] To evaluate the type of protein in the residue an ELISA analysiswas used to quantitate immunologically active casein and whey protein.In the casein and whey ELISA analysis, samples were tested undiluted,and at subsequent 4 fold dilutions up to and including a dilution of 1to 262,144. The enzyme-linked immunosorbent assay (ELISA) methodutilized is described in the article by Cordle et al. (Evaluation of theImmunogenicity of protein hydrolysate formulas using laboratory animalhyperimmunization. PEDIATRIC ALLERGY AND IMMUNOLOGY (5) p.14-19,1994).The ELISA assay is designed for the detection and quantitation ofimmunologically active casein or whey using rabbit anti-whey antibodyand rabbit anti-casein antibody.

[0096] This assay measures only the immunologically active casein andwhey protein components in the residue. Results of the ELISA analysesfor two separate reconstitutions (A and B) of each powdered human milkfortifier are shown in Table 4 below. TABLE 4 ELISA analysis of residuefor immunologically active casein and whey protein Sample Total mgcasein Total mg whey 2% milk control 1.31 0.31 IC - A 2.55 (1.24) 1.89(1.58) IC - B 3.49 (2.18) 2.32 (2.01) SC - A 4.98 (3.67) 3.51 (3.20)SC - B 5.77 (4.46) 4.94 (4.63)

[0097] All samples left immunologically active casein and whey proteinon the walls of the reconstitution container. However, the SC fortifierpowder left 137% more casein and 118% more whey protein behind onaverage than the IC fortifier powder of the instant invention whencorrected for contribution of the control.

[0098] To evaluate the amount of protein lost in the residue, the amountof total protein in each sample must be determined. First, the quantityof protein in solution is determined. The solution which was poured fromthe graduated cylinder in the preparation step above was analyzed by theTECATOR KJELTEC AUTO 1030 system for nitrogen to determine the quantityof protein that did not adhere to the reconstitution container (theprotein in solution).

[0099] The TECATOR KJELTEC AUTO 1030 system (Perstorp Analytical, Inc.)is an integrated, semi-automated nitrogen analyzer which uses anadaptation of the classical acid digestion/ammonia distillationprocedure first described by Johann Kjeldahl in 1883. The operatingmanuals for the Kjeltec Auto 1030 System were followed to determine theamount of nitrogen in the samples.

[0100] The amount of protein in the sample is calculated, based on thenitrogen concentration. The nitrogen concentrations in various proteinsare known, and empirically-determined conversion factors is used toconvert from % nitrogen to % protein. For example, milk proteinscontain, on the average, 15.67% nitrogen; thus, for milk proteins,

% Protein=% Nitrogen×(100% Protein/15.67% Nitrogen)

[0101] or

% Protein=% Nitrogen×6.38

[0102] Using the percent protein data and the known reconstitutionvolume (25 mL) the total protein that did not adhere to the sides of thecontainer can be calculated. The amount of protein that did not adhereto the sides of the reconstitution container for each sample is shown inTable 5 below. TABLE 5 Amount of total protein that did not adhere tothe sides of the reconstitution container Sample mg protein/ml total mgprotein 2% milk control 34.2   855 IC - A 43.4 (9.2) 1,085 (230) IC - B43.4 (9.2) 1,085 (230) SC - A 39.7 (5.5) 992.5 (137.5) SC - B 39.6 (5.4)  990 (135)

[0103] The total mg of protein in solution for the SC sample is 136.25mg on average and the total mg of protein in solution for the IC sampleis 230 mg on average. These differences are expected as each fortifierpowder contributes different levels of protein to the milk solution.These values are utilized below to calculate the percent total proteinlost as residue on the side of the reconstitution container.

[0104] The protein residue was tested for immunologicaly active whey andcasein above. However, there could be additional protein present in theresidue that would be detected. So, the total protein concentration ofthe residue was measured by the isoabsorbance method described in apublication by Ehresmann et al. (Spectrophotometric determination ofprotein concentration in cell extracts containing tRNA's and rRNA's,ANALYTICAL BIOCHEMISTRY (54) p. 454-463, 1973). A single reconstitutionof each of the formulas and the milk control were prepared as previouslydescribed, except that the residue was dissolved in 10.0 mL of PBS.Isoabsorbance measures all protein present, and cannot differentiateamong protein components. The results are shown in the Table 6 below.TABLE 6 Amount of total protein in the residue of the reconstitutioncontainer Sample mg protein/ml total mg protein 2% milk control 0.45 4.5 IC  0.9 (0.45)   9 (4.5) SC 1.37 (0.92) 13.7 (9.2)

[0105] By this technique, additional protein that was not detected bythe ELISA method was found in the residue. The SC fortifier powdersample left 104% more total protein behind than the IC fortifier powderof the instant invention when corrected for contribution of control.

[0106] Using the estimate of the protein which did not adhere to thewalls of the reconstitution container above, and the value for the totalprotein measured in the residue, a percent of protein lost can begenerated for each sample. Table 7 lists the total protein lost in theresidue clinging to the reconstitution container. TABLE 7 Total proteinlost in the reconstitution container Sample Residue/total protein X 100Protein lost in residue 2% milk control  (4.5/855 + 4.5) 100 0.52% IC -A (4.5/230 + 4.5)100 1.9% IC - B (4.5/230 + 4.5)100 1.9% SC - A(9.2/137.5 + 9.2)100   6.3% SC - B (9.2/135 + 9.2)100 6.4%

[0107] The SC fortifier powder lost 6% or 234% more protein in theresidue clinging to the sides of the container on average than the ICfortifier powder of the instant invention when corrected forcontribution of control. Clearly, the presence of soluble calciumdenatures the protein causing it to cling to the sides of the mixingcontainers and negatively impacts the amount of protein delivered to thepremature infant.

[0108] As discussed above, protein is crucial for growth of prematureinfants. The lose of protein helps explain the difference in growthobserved in preterm infants supplemented with the commercially availablepowdered human milk fortifier containing soluble calcium and thepowdered human milk fortifier of the instant invention containinginsoluble calcium. (See Experiment III and IV)

Experiment III

[0109] The primary objective of the study was to demonstrate that thefortifier powder of the instant invention added to human milk (HM) wouldsupport acceptable growth in preterm infants. A second objective was toevaluate the serum biochemistries (ie, protein status, calcium, alkalinephosphatase), tolerance, clinical problems, and morbidity of prematureinfants consuming the nutritional module. Another secondary objectivewas to compare the nutritional powder of the instant invention to acommercial fortifier powder that has been in use for a number of years.

[0110] An intent-to-treat, prospective, randomized, double-blindedmulticenter study was conducted to evaluate preterm infants receivingpreterm milk supplemented with either a commercially available powderedhuman milk fortifier (Enfamil® Human Milk Fortifier, control) or thefortifier powder of the current invention (experimental) at everyfeeding. Subjects were enrolled and randomized to each fortifier powderprior to 21 days of life. Study Day 1 was when fortification of thestudy fortifier powder had begun and the subject reached an intake of atleast 100 mL/kg/day. Anthropometric indices, serum biochemistries,intake, tolerance, and morbidity data were assessed. Each infant wasstudied until hospital discharge; only anthropometric variables (weight,length, and head circumference) were collected after Study Day 29.

[0111] Premature infants were recruited from neonatal intensive careunits that had agreed to collaborate with study investigators located inSalt Lake City, Utah; Houston, Tex.; Indianapolis, Ind.; Kansas City,Mo.; Louisville, Ky.; and Omaha, Nebr.

[0112] Single, twin, or triplet infants born ≦33 weeks gestational age,with appropriate weight for gestational age, and weighing ≦1600 g wereeligible to participate. One-hundred and forty-four infants wererandomized to either control or experimental; 70 preterm infants wererandomized to the control group and 74 preterm infants were randomizedto the experimental group. The randomization was proportional for birthweight (<1100 g and ≦1100 g) and gender.

[0113] The nutrient content of the two powder fortifiers added to humanmilk is listed in Table 8. TABLE 8 Nutrient Content of Human Milk,control and experimental fortifiers Preterm experimental‡ Milk-Mature*control† (per 3.6 g) Nutrients (per 100 mL) (per 3.8 g) Batch 1 Batch 2Energy, kcal/ml 67 14 13.8 14 Protein, g 1.4 0.7 1.0 1.0 source maturehuman whey protein whey protein whey protein milk concentrate/concentrate/ concentrate/ sodium nonfat dry milk nonfat dry milkcaseinate Fat, g 3.9 <0.1 0.41 0.37 source mature human none added MCToil MCT oil milk Carbohydrate, g 6.6 2.7 1.6 1.7 source lactose cornsyrup corn syrup corn syrup solids solids solids Vitamins A, IU 389 950640 763 D, IU 2.0 210 134 145 E, IU 1.0 4.6 3.4 3.5 K, mcg 0.21§ 4.408.50 8.60 Thiamin (B-1), mg 0.021§ 0.151 0.270 0.300 Riboflavin (B-2),mg 0.048 0.210 0.460 0.500 B-6, mg 0.015 0.114 0.230 0.240 B-12, mcg0.05§ 0.18 0.85 0.83 Niacin, mg 0.15§ 3.0 0.40 0.42 Folic acid, mcg 3.325 30 27 Pantothenic acid, mg 0.18§ 0.73 1.60 1.40 Biotin, mcg 0.4 2.730.0 33.0 C, mg 11 11.6 23.3 60.0 Minerals Calcium, mg 25 90 131 118Phosphorus, mg 13 45 68 67 Magnesium, mg 3.2 1.0 7.7 7.7 Zinc, mg 0.340.71 1.30 1.2 Manganese, mcg 0.6 4.7 9.2 12.0 Copper, mg 0.064 0.0620.200 0.200 Sodium, mg 25.0 7.0 17.5 17.5 Potassium, mg 57 15.6 72.072.0 Chloride, mg 55.0 17.7 40.0 40.0 Iron, mg 0.12 — 0.35 0.45Selenium, μg 2.3 — 1.4 0.99

[0114] The independent variables (treatments) were the control fortifierpowder and the experimental fortifier powder which were added to HM.Both fortifiers were provided in small packets in powdered form and wereadded to 25 mL HM.

[0115] The primary outcome variable was weight gain (g/kg/day) fromstudy day 1 to study day 29 or discharge, whichever came first.Secondary outcome variables were length gain (mm/day) and serumbiochemistries to evaluate protein status, electrolyte status, mineralhomeostasis, and vitamin A and E status. Serum biochemistries alsoincluded unscheduled laboratory results recorded in the medical chart.Tertiary variables included head circumference gain (mm/day), clinicalhistory, intake, tolerance, clinical problems/morbidity, respiratorystatus, antibiotic use, and the number of transfusions.

[0116] Mean total energy intakes during the study period was notdifferent between the two groups. Infants fed control fortified humanmilk received 118.0±2.2 kcal/kg/day while the infants fed experimentalfortified human milk received 118.0±1.6 kcal/kg/day. There was adifference in mean protein intake between the two groups. Infants fedcontrol fortified human milk received 3.1±0.1 g protein/kg/day whileinfants fed experimental fortified human milk received 3.5±0.1 gprotien/kg/day which is consistent with the slightly higher proteincontent of experimental fortifier powder.

[0117] There were consistent differences among infants in the twofortifier powder groups with respect to growth (weight, length, headcircumference) with the control group always growing more slowly. Thesignificant difference in weight gain for the primary analysis betweenthe groups was 2.6 g/kg/day (experimental>control; p<0.0005). Thesignificant difference in length gain for the primary analysis betweenthe groups was 0.2 mm/day (experimental>control; p<0.05). Although theprimary analysis for head circumference gain was not different betweenthe groups, there was a significant difference in head circumferencegain of 0.15 mm/day when analyzed from study day 1 to last fortifierpowder feeding (experimental>control; p<0.05). Furthermore, thedifferences in weight, length, and head circumference gains between thegroups were even greater in the evaluable group of infants than in theintent-to-treat group of infants. The significant differences in weight,length, and head circumference gains (p<0.0005; p<0.05; and p<0.001,respectively) for the primary analyses were 4.07 g/kg/day, 0.23 mm/day,and 0.30 mm/day, respectively, with the control group growing moreslowly.

[0118] The differences in serum biochemistries between the groups werealso very consistent between the intent-to-treat and evaluable groups.Of most clinical significance were the mean alkaline phosphatase andcalcium values. In the intent-to-treat group, mean alkaline phosphatasevalues among infants randomized to the experimental group appeared to behigher compared to the infants randomized to the control group (327 U/Lvs 272 U/L, respectively); however, mean values for both groups weredecreased by study day 29. In addition, mean alkaline phosphatase was inthe normal range for both groups. Results were similar in the evaluablegroup. In contrast, in the intent-to-treat group, the mean serum calciumvalues among infants fed control fortifier powder tended to be higheroverall than among infants fed experimental fortifier powder (p=0.069).As expected, in the evaluable group the overall differences were greaterwith mean calcium values of 11.2 mg/dL in the control group and 10.3mg/dL in the experimental group (p=0.016). A value greater than 11 mg/dLis considered in the upper range and is suggestive of hypercalcemia.

[0119] There were no significant differences between the groups inrespect to clinical problems or morbidity data. Overall tolerance toboth products was excellent. The only difference was in the episodes ofvomiting with the control group having a greater number (percent) ofdays with vomiting episodes than the experimental group (18% vs 11%;p<0.01). The control group also had a statistically greater number ofinfants who exited early from the study for reasons of gastric residualsand/or abdominal distention than did the experimental group (6 vs 0;p=0.012).

[0120] The primary findings of this study suggest that increases inprotein, the addition of fat and emulsifier and use of insoluble calciumin the fortifier powder of the instant invention results in dramaticimprovements in growth performance of low-birth-weight infants eventhough the caloric density has not been changed.

Experiment IV

[0121] The experiments above each describe the positive benefits ofusing insoluble calcium as the calcium source of choice in the fortifierof the instant invention. Less protein is left behind in the mixingcontainers which shows positive benefits in premature infant growth.However, bone mineralization is a concern with the use of insolublecalcium.

[0122] The objectives of this study were similar to those in ExperimentIll. A preliminary evaluation of mineral bioavailability, specificallycalcium, of the fortifier powder of the instant invention was added tothe protocol through documentation of forearm bone density of prematureinfants supplemented with the fortifier powder of the instant inventionwhich contains insoluble calcium. These forearm bone density values werecompared to those of premature infants supplemented with a commerciallyavailable fortifier powder which contains soluble calcium.

[0123] The protocol was as described in Experiment III. A prospective,randomized, double-blinded study was conducted to evaluate preterminfants receiving their own mother's milk supplemented with either acommercial powdered human milk fortifier containing soluble calcium(Enfamil® Human Milk Fortifier, control) or the fortifier powder of theinstant invention containing insoluble calcium (experimental) at everyfeeding. Anthropometric indices, serum biochemistries and forearm's bonedensity, fat and lean mass were studied at days 1,15 and 29. Infant'sforearm was scanned using the dual x-ray absorption method.

[0124] Single, twin, or triplet infants born ≦33 weeks gestational age,with appropriate weight for gestational age, and weighing ≦1500 g wereeligible to participate. Forty-three infants were randomized to eithercontrol or experimental.

[0125] On day one, there were no differences between control andexperimental groups in weight, length, head circumference or forearm'sbone density, fat and lean mass.

[0126] Mean total energy intakes during the study period were similar(110±15 vs 114±9 kcal/dg/d). However, there was a significant differencein weight gain between the groups, 18±2 vs 15±3 glkg/day(experimental>control; p=0.0003). There were no differences in length orhead circumference gains between the two groups and the serumbiochemistries were similar between the groups. By day 15, theexperimental group had a higher lean mass than controls 16±3 gm vs 14±3gm (p<0.05).

[0127] Bone mineral content (0.608±0.172 vs 0.631±0.175 g/cm) and fatmass (6.9±3.3 vs 7.5±3.7 gm) were similar during the study.

[0128] These findings suggest that the insoluble calcium of thefortifier powder of the instant invention was as available as thesoluble calcium of the control fortifier powder. Additionally, thegrowth findings of this study are consistent with the results from theprevious study. These findings suggest that the fortifier powder of theinstant invention provides higher weight gain in preterm infants than acommercial fortifier powder and suggest that the weight gain may be fromhigher lean mass gain.

[0129] The embodiments of the present invention may, of course, becarried out in other ways than those set forth herein without departingfrom the spirit and scope of the invention. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive and that all changes and equivalents also come within thedescription of the present invention.

We claim:
 1. A powdered human milk fortifier comprising: a. a proteincomponent present in a quantity of from about 25 wt/wt % to about 42wt/wt % of the powdered human milk fortifier, b. a fat component presentin a quantity of from about 5 wt/wt % to about 30 wt/wt % of thepowdered human milk fortifier wherein said fat component furthercomprises an emulsifier present in a quantity of from about 1.5 wt/wt %to about 5.0 wt/wt % of said fat component, and c. at least oneadditional nutrient selected from the group consisting of Vitamin A,Vitamin B₁, Vitamin B₂, Vitamin B₆, Vitamin B₁₂, Vitamin C, Vitamin D,Vitamin E, Vitamin K, Biotin, Folic Acid, Pantothenic Acid, Niacin,m-inositol, calcium, phosphorus, magnesium, zinc, manganese, copper,sodium, potassium, chloride, iron, selenium, chromium, molybdenum,carnitine and taurine, and wherein said calcium source is insoluble. 2.A method of providing supplemental nutrients to preterm infantscomprising adding the powdered human milk fortifier according to claim 1to human milk and administering a fortified human milk to a prematureinfant.
 3. A method for promoting growth of a premature infantcomprising administering fortified human milk to a premature infant,said fortified human milk further comprises: a. human milk, and b. thepowdered human milk fortifier according to claim
 1. 4. A unit dose ofpowdered human milk fortifier comprising: a. a container, and b. thepowdered human milk fortifier according to claim 1 present in an amountof from about 0.8 gm to about 5.0 gm per unit dose.
 5. A method forenhancing the emulsion stability of human milk comprising adding anemulsifier to said human milk wherein said emulsifier is present in aquantity of from about 0.36 wt/vol % to about 3.6 wt/vol % of the humanmilk.
 6. A method for preventing the denaturation of protein infortified human milk comprising the incorporation of an insolublecalcium source.
 7. A powdered human milk fortifier comprising: a) aprotein component present in a quantity of from about 28 wt/wt % toabout 55 wt/wt % of the powdered human milk fortifier; b) a fatcomponent present in a quantity of from about 1 wt/wt % to about 30wt/wt % of the powdered human milk fortifier; and c) at least one sourceof insoluble calcium.
 8. The powdered human milk fortifier according toclaim 7 in which: a) said protein component is present in the quantityof from about 28 wt/wt % to about 42 wt/wt %; and said fat component ispresent in the quantity of from about 1 wt/wt % to about 18 wt/wt %. 9.A method of providing supplemental nutrients to preterm infantscomprising adding the powdered human milk fortifier according to claim 7to human milk and administering a fortified human milk to a prematureinfant.
 10. A method for promoting growth of a premature infantcomprising administering fortified human milk to a premature infant,said fortified human milk comprises: a) human milk, and b) the powderedhuman milk fortifier according to claims
 7. 11. The method of providingsupplemental nutrients according to claim 9 wherein about 0.5 gm toabout 10 gm of powdered human milk fortifier is administered per day.12. A fortified human milk comprising: a) human milk, and b) from about0.5 wt/vol % to about 10.0 wt/vol % of a powdered human milk fortifierwhich comprises: i) a protein component present in a quantity of fromabout 27 wt/wt % to about 42 wt/wt % of the powdered human milkfortifier; ii) a fat component present in a quantity of from about 1wt/wt % to about 30 wt/wt % of the powdered human milk fortifier; andiii) at least one source of insoluble calcium.
 13. A fortified humanmilk comprising: a) human milk, and b) from about 0.5 wt/vol % to about10.0 wt/vol % of a powdered human milk fortifier which comprises: i. aprotein component present in a quantity of from about 27 wt/wt % toabout 42 wt/wt % of the powdered human milk fortifier; ii. a fatcomponent present in a quantity of from about 1 wt/wt % to about 30wt/wt % of the powdered human milk fortifier.
 14. A powdered human milkfortifier comprising: a) a protein component present in a quantity offrom about 25 wt/wt % to about 55 wt/wt % of the powdered human milkfortifier; b) a fat component present in a quantity of from about 1wt/wt % to about 30 wt/wt % of the powdered human milk fortifier; and;c) at least one source of insoluble calcium.
 15. The powdered human milkfortifier according to claim 14 in which said fat component is presentin the quantity of about 5 wt/wt % to about 30 wt/wt %
 16. The powderedhuman milk fortifer according to claim 14 in which: a) said proteincomponent is present in the quantity of from about 27 wt/wt % to about36 wt/wt %, and b) said fat component is present in the quantity of fromabout 1 wt/wt % to about 20 wt/wt %.
 17. A method of providingsupplemental nutrients to preterm infants comprising adding the powderedhuman milk fortifier according to claim 14 to human milk andadministering a fortified human milk to a premature infant.
 18. A methodfor promoting growth of a premature infant comprising administeringfortified human milk to a premature infant, said fortified human milkcomprises: a) human milk, and b) the powdered human milk fortifieraccording to claim 15.